Hepatitis b antiviral agents

ABSTRACT

Provided herein are compounds useful for the treatment of HBV infection in man.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 61/778,144, filed Mar. 12, 2013, the content of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Chronic hepatitis B virus (HBV) infection is a significant global healthproblem, affecting over 5% of the world population (over 350 millionpeople worldwide and 1.25 million individuals in the U.S.).

Despite the availability of a prophylactic HBV vaccine, the burden ofchronic HBV infection continues to be a significant unmet worldwidemedical problem, due to suboptimal treatment options and sustained ratesof new infections in most parts of the developing world. Currenttreatments do not provide a cure and are limited to only two classes ofagents (interferon and nucleoside analogues/inhibitors of the viralpolymerase); drug resistance, low efficacy, and tolerability issueslimit their impact. The low cure rates of HBV are attributed at least inpart to the presence and persistence of covalently closed circular DNA(cccDNA) in the nucleus of infected hepatocytes. However, persistentsuppression of HBV DNA slows liver disease progression and helps toprevent hepatocellular carcinoma. Current therapy goals for HBV-infectedpatients are directed to reducing serum HBV DNA to low or undetectablelevels, and to ultimately reducing or preventing the development ofcirrhosis and hepatocellular carcinoma.

There is a need in the art for novel therapeutic agents that treat,ameliorate or prevent HBV infection. Administration of these therapeuticagents to an HBV infected patient, either as monotherapy or incombination with other HBV treatments or ancillary treatments, will leadto significantly improved prognosis, diminished progression of thedisease, and enhanced seroconversion rates.

SUMMARY OF THE INVENTION

Provided herein are compounds useful for the treatment of HBV infectionin man.

Accordingly, in one aspect, provided herein is a compound of Formula I,II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII, or pharmaceuticallyacceptable salts thereof. In one embodiment, provided herein is apharmaceutical composition further comprising a compound of Formula I,II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII, or a pharmaceuticallyacceptable salt thereof, at least one pharmaceutically acceptablecarrier.

In one aspect, provided herein is a method of treating, eradicating,reducing, slowing, or inhibiting an HBV infection in an individual inneed thereof, comprising administering to the individual atherapeutically effective amount of a compound of Formula I, II, III,IV, V, VI, VII, VIII, IX, X, XI, or XII, or a pharmaceuticallyacceptable salt thereof.

In another aspect, provided herein is a method of reducing the viralload associated with an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of Formula I, II, III, IV, V, VI, VII, VIII, IX, X,XI, or XII, or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a method of reducingreoccurrence of an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of Formula I, II, III, IV, V, VI, VII, VIII, IX, X,XI, or XII, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of reducing an adversephysiological impact of an HBV infection in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of a compound of Formula I, II, III, IV, V, VI, VII,VIII, IX, X, XI, or XII, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of inducing remission ofhepatic injury from an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of Formula I, II, III, IV, V, VI, VII, VIII, IX, X,XI, or XII, or a pharmaceutically acceptable salt thereof.

In still another aspect, provided herein is a method of reducing thephysiological impact of long-term antiviral therapy for HBV infection inan individual in need thereof, comprising administering to theindividual a therapeutically effective amount of Formula I, II, III, IV,V, VI, VII, VIII, IX, X, XI, or XII, or a pharmaceutically acceptablesalt thereof.

In another aspect, provided herein is a method of prophylacticallytreating an HBV infection in an individual in need thereof, wherein theindividual is afflicted with a latent HBV infection, comprisingadministering to the individual a therapeutically effective amount of acompound of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII,or a pharmaceutically acceptable salt thereof.

In one embodiment of any of the methods described above, the methodsfurther comprise administering to the individual at least one additionaltherapeutic agent selected from the group consisting of a HBV polymeraseinhibitor, interferon, pegylated interferon, viral entry inhibitor,viral maturation inhibitor, literature-described capsid assemblymodulator, reverse transcriptase inhibitor, a TLR-agonist, and agents ofdistinct or unknown mechanism, and a combination thereof.

In one embodiment, the pegylated interferon is pegylated interferonalpha (IFN-α) (e.g., PEGASYS®), pegylated interferon lambda (IFN-λ), orpegylated interferon gamma (IFN-γ).

In another embodiment, the reverse transcriptase inhibitor is at leastone of Zidovudine, Didanosine, Zalcitabine, ddA, Stavudine, Lamivudine,Abacavir, Emtricitabine, Entecavir, Apricitabine, Atevirapine,ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir,valganciclovir, Tenofovir, Adefovir, PMPA, cidofovir, Efavirenz,Nevirapine, Delavirdine, or Etravirine.

In still another embodiment, the compound and the at least oneadditional therapeutic agent are co-formulated. In another embodiment,the compound and the at least one additional therapeutic agent areco-administered.

In one embodiment of the combination therapies, administering thecompound of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII,or a pharmaceutically acceptable salt thereof, allows for administeringthe at least one additional therapeutic agent at a lower dose orfrequency as compared to the administering of the at least oneadditional therapeutic agent alone that is required to achieve similarresults in prophylactically treating an HBV infection in an individualin need thereof.

In another embodiment of these methods, before administering thetherapeutically effective amount of the compound of Formula I, II, III,IV, V, VI, VII, VIII, IX, X, XI, or XII, or a pharmaceuticallyacceptable salt thereof, the individual is known to be refractory to acompound selected from the group consisting of a HBV polymeraseinhibitor, interferon, pegylated interferon, viral entry inhibitor,viral maturation inhibitor, distinct capsid assembly modulator,antiviral compounds of distinct or unknown mechanism, and combinationthereof.

In still another embodiment, administering of the compound of Formula I,II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII, or a pharmaceuticallyacceptable salt thereof, reduces viral load in the individual to agreater extent compared to the administering of a compound selected fromthe group consisting of a HBV polymerase inhibitor, interferon,pegylated interferon, viral entry inhibitor, viral maturation inhibitor,distinct capsid assembly modulator, antiviral compounds of distinct orunknown mechanism, and combination thereof.

In another embodiment, administering of the compound of Formula I, II,III, IV, V, VI, VII, VIII, IX, X, XI, or XII, or a pharmaceuticallyacceptable salt thereof, causes a lower incidence of viral mutationand/or viral resistance than the administering of a compound selectedfrom the group consisting of a HBV polymerase inhibitor, interferon,pegylated interferon, viral entry inhibitor, viral maturation inhibitor,distinct capsid assembly modulator, antiviral compounds of distinct orunknown mechanism, and combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compounds that are useful in the treatment andprevention of HBV in man. In a non-limiting aspect, these compoundsmodulate and/or disrupt HBV assembly by interacting with HBV capsid toafford defective viral particles with greatly reduced virulence. Thecompounds of the invention have potent antiviral activity, exhibitfavorable metabolic, tissue distribution, safety and pharmaceuticalprofiles, and are suitable for use in man.

The HBV capsid protein plays essential functions during the viral lifecycle. HBV capsid/core proteins form metastable viral particles orprotein shells that protect the viral genome during intercellularpassage, and also play a central role in viral replication processes,including genome encapsidation, genome replication, and virionmorphogenesis and egress. Capsid structures also respond toenvironmental cues to allow un-coating after viral entry. Consistently,proper capsid assembly has been found to be critical for viralinfectivity.

The crucial function of HBV capsid proteins imposes stringentevolutionary constraints on the viral capsid protein sequence, leadingto the observed low sequence variability and high conservation.Consistently, mutations in HBV capsid that disrupt its assembly arelethal, and mutations that perturb capsid stability severely attenuateviral replication. The more conserved a drug target is, the fewerreplication-competent resistance mutations are acquired by patients.

Indeed, natural mutations in HBV capsid for chronically infectedpatients accumulate in only four out of 183 residues in the full lengthprotein. Thus, HBV capsid assembly inhibitors may elicit lower drugresistance emergence rates relative to existing HBV antivirals. Further,drug therapy that targets HBV capsid could be less prone todrug-resistant mutations when compared to drugs that target traditionalNA enzyme active sites. Reports describing compounds that bind viralcapsids and inhibit replication of HIV, rhinovirus and HBV providestrong pharmacological proof of concept for viral capsid proteins asantiviral drug targets.

In one aspect, the compounds of the invention are useful in HBVtreatment by disrupting, accelerating, reducing, delaying and/orinhibiting normal viral capsid assembly and/or disassembly of immatureor mature particles, thereby inducing aberrant capsid morphology andleading to antiviral effects such as disruption of virion assemblyand/or disassembly, virion maturation, and/or virus egress. In oneembodiment, a disruptor of capsid assembly interacts with mature orimmature viral capsid to perturb the stability of the capsid, thusaffecting assembly and/or disassembly. In another embodiment, adisruptor of capsid assembly perturbs protein folding and/or saltbridges required for stability, function and/or normal morphology of theviral capsid, thereby disrupting and/or accelerating capsid assemblyand/or disassembly. In yet another embodiment, the compounds of theinvention bind capsid and alter metabolism of cellular polyproteins andprecursors, leading to abnormal accumulation of protein monomers and/oroligomers and/or abnormal particles, which causes cellular toxicity anddeath of infected cells. In another embodiment, the compounds of theinvention cause failure of the formation of capsid of optimal stability,affecting efficient uncoating and/or disassembly of viruses (e.g.,during infectivity).

In one embodiment, the compounds of the invention disrupt and/oraccelerate capsid assembly and/or disassembly when the capsid protein isimmature. In another embodiment, the compounds of the invention disruptand/or accelerate capsid assembly and/or disassembly when the capsidprotein is mature. In yet another embodiment, the compounds of theinvention disrupt and/or accelerate capsid assembly and/or disassemblyduring vial infectivity. In yet another embodiment, the disruptionand/or acceleration of capsid assembly and/or disassembly attenuates HBVviral infectivity and/or reduces viral load. In yet another embodiment,disruption, acceleration, inhibition, delay and/or reduction of capsidassembly and/or disassembly eradicates the virus from the host organism.In yet another embodiment, eradication of the HBV from a hostadvantageously obviates the need for chronic long-term therapy and/orreduces the duration of long-term therapy.

In one embodiment, the compounds described herein are suitable formonotherapy and are effective against natural or native HBV strains andagainst HBV strains resistant to currently known drugs. In anotherembodiment, the compounds described herein are suitable for use incombination therapy.

In another embodiment, the compounds of the invention can be used inmethods of modulating (e.g., inhibit, disrupt or accelerate) theactivity of HBV cccDNA. In yet another embodiment, the compounds of theinvention can be used in methods of diminishing or preventing theformation of HBV cccDNA.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and peptide chemistryare those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e. to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

As used herein, the term “capsid assembly modulator” refers to acompound that disrupts and/or accelerates and/or inhibits and/or hindersand/or delays and or reduces and/or modifies normal capsid assembly(e.g., during maturation) and/or normal capsid disassembly (e.g., duringinfectivity) and/or perturbs capsid stability, thereby inducing aberrantcapsid morphology and function. In one embodiment, a capsid assemblymodulator accelerates capsid assembly and/or disassembly, therebyinducing aberrant capsid morphology. In another embodiment, a capsidassembly modulator interacts (e.g. binds at an active site, binds at anallosteric site, modifies and/or hinders folding and the like) with themajor capsid assembly protein (CA), thereby disrupting capsid assemblyand/or disassembly. In yet another embodiment, a capsid assemblymodulator causes a perturbation in structure and/or function of CA(e.g., ability of CA to assemble, disassemble, bind to a substrate, foldinto a suitable conformation, or the like), which attenuates viralinfectivity and/or is lethal to the virus.

As used herein, the term “literature-described capsid assemblymodulator” refers a capsid assembly modulator that is not a compound ofthe present invention.

As used herein, the term “treatment” or “treating,” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has HBV infection, a symptom ofHBV infection or the potential to develop HBV infection, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect HBV infection, the symptoms of HBV infection or thepotential to develop HBV infection. Such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the term “patient,” “individual” or “subject” refers toa human or a non-human mammal. Non-human mammals include, for example,livestock and pets, such as ovine, bovine, porcine, canine, feline andmurine mammals. Preferably, the patient, subject or individual is human.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compounds prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic,propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric,succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic.Appropriate organic acids may be selected, for example, from aliphatic,aromatic, carboxylic and sulfonic classes of organic acids, examples ofwhich are formic, acetic, propionic, succinic, camphorsulfonic, citric,fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric,para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic(besylate), stearic, sulfanilic, alginic, galacturonic, and the like.Furthermore, pharmaceutically acceptable salts include, by way ofnon-limiting example, alkaline earth metal salts (e.g., calcium ormagnesium), alkali metal salts (e.g., sodium-dependent or potassium),and ammonium salts.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

As used herein, “pharmaceutically acceptable carrier” also includes anyand all coatings, antibacterial and antifungal agents, and absorptiondelaying agents, and the like that are compatible with the activity ofthe compound useful within the invention, and are physiologicallyacceptable to the patient. Supplementary active compounds may also beincorporated into the compositions. The “pharmaceutically acceptablecarrier” may further include a pharmaceutically acceptable salt of thecompound useful within the invention. Other additional ingredients thatmay be included in the pharmaceutical compositions used in the practiceof the invention are known in the art and described, for example inRemington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co.,1985, Easton, Pa.), which is incorporated herein by reference.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient orsubject. Multiple techniques of administering a compound exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e., C₁₋₆means one to six carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “substituted alkyl” means alkyl as definedabove, substituted by one, two or three substituents selected from thegroup consisting of halogen, —OH, alkoxy, —NH₂, —N(CH₃)₂, —C(═O)OH,trifluoromethyl, —C≡N, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂, —SO₂NH₂,—C(═NH)NH₂, and —NO₂, preferably containing one or two substituentsselected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and—C(═O)OH, more preferably selected from halogen, alkoxy and —OH.Examples of substituted alkyls include, but are not limited to,2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃. Preferred heteroalkyl groups have1-10 carbons.

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” or “carbocyclyl” refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atomsforming the ring (i.e., skeletal atoms) is a carbon atom. In oneembodiment, the cycloalkyl group is saturated or partially unsaturated.In another embodiment, the cycloalkyl group is fused with an aromaticring. Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon carbondouble bond or one carbon carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, S and N. In one embodiment, each heterocycloalkyl grouphas from 4 to 10 atoms in its ring system, with the proviso that thering of said group does not contain two adjacent O or S atoms. Inanother embodiment, the heterocycloalkyl group is fused with an aromaticring. In one embodiment, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Inone embodiment, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine. Other non-limiting examples ofheterocycloalkyl groups are:

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e., having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl. Preferred examples arephenyl and naphthyl, most preferred is phenyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In one embodiment, the substituents vary in number between one and four.In another embodiment, the substituents vary in number between one andthree. In yet another embodiment, the substituents vary in numberbetween one and two.

Compounds of the Invention

The present invention relates to the discovery of compounds that areuseful in the treatment and prevention of HBV in man. In one aspect, thecompounds of the invention are useful in HBV treatment by disrupting,accelerating, reducing delaying and/or inhibiting normal viral capsidassembly and/or disassembly of immature or mature particles, therebyinducing aberrant capsid morphology and leading to antiviral effectssuch as disruption of virion assembly and/or disassembly and/or virionmaturation, and/or virus egress.

The capsid assembly disruptors disclosed herein may be used asmonotherapy and/or in novel cross-class combination regimens fortreating HBV infection in man. Combination therapy with drugs exhibitingdifferent mechanism of action (MOA) that act at different steps in thevirus life cycle may deliver greater efficacy due to additive orsynergistic antiviral effects. Clinically evaluated HIV treatmentregimens have shown that combination therapy improves the efficacy ofviral load reduction, and dramatically reduces emergence of antiviralresistance. Combination therapy for the treatment of Hepatitis C (HCV)virus infection has also resulted in significant improvement insustained antiviral response and eradication rates. Thus, use of the HBVcapsid assembly inhibitors of the present invention in combination with,for example, NA drugs, is likely to deliver a more profound antiviraleffect and greater disease eradication rates than current standards ofcare.

Capsid assembly plays a central role in HBV genome replication. HBVpolymerase binds pre-genomic HBV RNA (pgRNA), and pgRNA encapsidationmust occur prior to HBV DNA synthesis. Moreover, it is well establishedthat nuclear accumulation of the cccDNA replication intermediate, whichis responsible for maintenance of chronic HBV replication in thepresence of nucleoside suppressive therapy, requires the capsid forshuttling HBV DNA to the nuclei. Therefore, the HBV capsid assemblydisruptors of the invention have the potential to increase HBVeradication rates through synergistic or additive suppression of viralgenome replication and to further reduce accumulation of cccDNA whenused alone or in combination with existing nucleoside drugs. The capsidassembly disruptors of the present invention may also alter normal coreprotein degradation, potentially leading to altered MHC-1 antigenpresentation, which may in turn increase seroconversion/eradicationrates through immuno-stimulatory activity, more effectively clearinginfected cells.

In one aspect, drug resistance poses a major threat to current therapiesfor chronic HBV infection, and cross-class combination therapy is aproven strategy for delaying emergence of drug resistance strains. Thecapsid assembly disruptors of the present invention can, whenadministered alone or in combination with other HBV therapy, offerenhanced drug resistant profiles and improved management of chronic HBV.

The compounds useful within the invention may be synthesized usingtechniques well-known in the art of organic synthesis. The startingmaterials and intermediates required for the synthesis may be obtainedfrom commercial sources or synthesized according to methods known tothose skilled in the art.

In one aspect, the compound of the invention is a compound of Formula I,

or a pharmaceutically acceptable salt thereof,

wherein:

Ring A is selected from:

wherein:

G is N, and Q is CH₂ or NR³, or

G is NR³, and Q is CH or N, or

G is CH, and Q is NR³, or

G is CH₂, and Q is N; and

represents a single or double bond;

T is selected from —C(O)—, —NH(C(O))— and —S(O)₂—;

V is selected from —C(O)— and —S(O)₂—;

R¹ in each occurrence is independently selected from halo, —CN,C₁₋₆alkyl, —OC₁₋₆ alkyl, and —NO₂, wherein the C₁₋₆alkyl is optionallysubstituted with one to three halo;

R² in each occurrence is independently selected from hydrogen, halo, andC₁₋₆alkyl;

R³ is selected from H and C₁₋₆ alkyl;

R⁴ in each occurrence is independently selected from halo, C₁₋₆alkyl,monocyclic 5 to 7-membered carbocyclyl, monocyclic 5 to 7-memberedheterocyclyl, —OR^(4a), —SR^(4a), and —N(R^(4a))₂, wherein theC₁₋₆alkyl, monocyclic 5 to 7-membered carbocyclyl, and monocyclic 5 to7-membered heterocyclyl are optionally substituted with one or more R⁴⁰;

R^(4a) in each occurrence is independently selected from H, C₁₋₆alkyl,monocyclic 5 to 7-membered carbocyclyl, and monocyclic 5 to 7-memberedheterocyclyl, wherein said C₁₋₆alkyl, monocyclic 5 to 7-memberedcarbocyclyl, and monocyclic 5 to 7-membered heterocyclyl, are optionallyand independently substituted with one or more R⁴⁰;

R⁵ is selected from H and C₁₋₆alkyl;

R⁴⁰ in each occurrence is independently selected from halo, —CN,C₁₋₆alkyl, monocyclic 5 to 7-membered carbocyclyl, monocyclic 5 to7-membered heterocyclyl, —OR^(40a), —SR^(40a), —N(R^(40a))₂, —NO₂,—C(O)R^(40b), and —C(O)₂R^(40a);

-   -   R^(40a) in each occurrence is independently selected from H,        C₁₋₆alkyl, monocyclic 5 to 7-membered carbocyclyl, and        monocyclic 5 to 7-membered heterocyclyl;

R^(40b) in each occurrence is independently selected from C₁₋₆alkyl,monocyclic 5 to 7-membered carbocyclyl, and monocyclic 5 to 7-memberedheterocyclyl;

w is 0, 1, 2, or 3;

x is 0 or 1; and

y is 0, 1, or 2.

In one embodiment of Formula I, R⁴ in each occurrence is independentlyselected from halo, C₁₋₆alkyl, and —OR^(4a), wherein the C₁₋₆alkyl isoptionally substituted with one or more halo; and R^(4a) in eachoccurrence is independently selected from H or C₁₋₆alkyl.

In one embodiment, the compound of Formula I is a compound of FormulaII:

or a pharmaceutically acceptable salt thereof,

wherein:

Ring A is selected from:

and

Q is CH or N, or

Ring A is

and Q is CH₂ or NR³.

In one embodiment of Formulas I or II, R¹ is halo.

In another embodiment of Formulas I or II, R¹ in each occurrence isindependently selected from fluorine and chlorine, R³ in each occurrenceis independently selected from H and methyl, R⁴ is selected from H andmethyl, and w is 0, 2, or 3.

In another embodiment of Formulas I or II, R¹ in each occurrence isindependently selected from fluorine and chlorine, R³ is H, R⁴ is H, andw is 0, 2, or 3.

In one embodiment, the compound of Formulas I or II is a compound ofFormula III:

or a pharmaceutically acceptable salt thereof.

In one embodiment of Formula III, R¹ in each occurrence is independentlyselected from fluorine and chlorine, R⁴ is selected from H and methyl,and w is 0, 2, or 3. In another embodiment of Formula III, R⁴ in eachoccurrence is independently selected from halo, C₁₋₆alkyl, and —OR^(4a),wherein the C₁₋₆alkyl is optionally substituted with one or more halo;and R^(4a) in each occurrence is independently selected from H orC₁₋₆alkyl.

In another embodiment, the compound of Formula III is a compound ofFormula IV:

or a pharmaceutically acceptable salt thereof.

In one embodiment of Formula IV, R¹ in each occurrence is independentlyselected from fluorine and chlorine, R⁴ is selected from H and methyl,and w is 0, 2, or 3. In another embodiment of Formula IV, R⁴ in eachoccurrence is independently selected from halo, C₁₋₆alkyl, and —OR^(4a),wherein the C₁₋₆alkyl is optionally substituted with one or more halo;and R^(4a) in each occurrence is independently selected from H orC₁₋₆alkyl.

In one embodiment, the compound of Formulas I or II is a compound ofFormula V:

or a pharmaceutically acceptable salt thereof.

In one embodiment of Formula V, R¹ in each occurrence is independentlyselected from fluorine and chlorine, R³ in each occurrence isindependently selected from H and methyl, R⁴ is selected from H andmethyl, and w is 0, 2, or 3. In another embodiment of Formula V, R⁴ ineach occurrence is independently selected from halo, C₁₋₆alkyl, and—OR^(4a), wherein the C₁₋₆alkyl is optionally substituted with one ormore halo; and R^(4a) in each occurrence is independently selected fromH or C₁₋₆alkyl.

In another embodiment, the compound of Formula V is a compound ofFormula VI:

or a pharmaceutically acceptable salt thereof.

In one embodiment of Formula VI, R¹ in each occurrence is independentlyselected from fluorine and chlorine, R³ in each occurrence isindependently selected from H and methyl, R⁴ is selected from H andmethyl, and w is 0, 2, or 3. In another embodiment of Formula VI, R⁴ ineach occurrence is independently selected from halo, C₁₋₆alkyl, and—OR^(4a), wherein the C₁₋₆alkyl is optionally substituted with one ormore halo; and R^(4a) in each occurrence is independently selected fromH or C₁₋₆alkyl.

In yet another embodiment, the compound of Formulas I or II is acompound of Formula VII:

or a pharmaceutically acceptable salt thereof.

In one embodiment of Formula VII, R¹ in each occurrence is independentlyselected from fluorine and chlorine, R³ in each occurrence isindependently selected from H and methyl, R⁴ is selected from H andmethyl, and w is 0, 2, or 3. In another embodiment of Formula VII, R⁴ ineach occurrence is independently selected from halo, C₁₋₆alkyl, and—OR^(4a), wherein the C₁₋₆alkyl is optionally substituted with one ormore halo; and R^(4a) in each occurrence is independently selected fromH or C₁₋₆alkyl.

In another embodiment, the compound of Formula VII is a compound ofFormula VIII:

or a pharmaceutically acceptable salt thereof.

In one embodiment of Formula VIII, R¹ in each occurrence isindependently selected from fluorine and chlorine, R³ in each occurrenceis independently selected from H and methyl, R⁴ is selected from H andmethyl, and w is 0, 2, or 3. In another embodiment of Formula VIII, R⁴in each occurrence is independently selected from halo, C₁₋₆alkyl, and—OR^(4a), wherein the C₁₋₆alkyl is optionally substituted with one ormore halo; and R^(4a) in each occurrence is independently selected fromH or C₁₋₆alkyl.

In another aspect, the compound of the invention is a compound ofFormula IX:

or a pharmaceutically acceptable salt thereof,

wherein:

Ring A is selected from:

wherein:

G is N, and Q is CH₂ or NR³, or

G is NR³, and Q is CH or N, or

G is CH, and Q is NR³, or

G is CH₂, and Q is N; and

represents a single or double bond;

T is selected from —C(O)—, —NH(C(O))—, and —S(O)₂—;

V is selected from —C(O)— and —S(O)₂—;

R¹ in each occurrence is independently selected from halo, —CN,C₁₋₆alkyl, —OC₁₋₆alkyl, and —NO₂, wherein the C₁₋₆alkyl is optionallysubstituted with one to three halo;

R² in each occurrence is independently selected from hydrogen, halo, andC₁₋₆alkyl;

R³ is selected from H and C₁₋₆alkyl;

R⁶ is H or C₁₋₆ alkyl;

R⁷ is H or C₁₋₆ alkyl;

n is 1, 2, 3, 4, 5, or 6; and

w is 0, 1, 2, or 3.

In one embodiment, the compound of Formula IX is a compound of FormulaX:

or a pharmaceutically acceptable salt thereof,

wherein:

Ring A is selected from:

and Q is CH or N, or

Ring A is

and Q is CH₂ or NR³.

In a further aspect, the compound of the invention is a compound ofFormula XI:

or a pharmaceutically acceptable salt thereof,

wherein:

Ring A is selected from:

wherein:

G is N, and Q is CH₂ or NR³, or

G is NR³, and Q is CH or N, or

G is CH, and Q is NR³, or

G is CH₂, and Q is N; and

represents a single or double bond;

T is selected from —C(O)—, —NH(C(O))—, and —S(O)₂—;

V is selected from —C(O)— and —S(O)₂—;

R¹ in each occurrence is independently selected from halo, —CN,C₁₋₆alkyl, —OC₁₋₆alkyl, and —NO₂, wherein the C₁₋₆alkyl is optionallysubstituted with one to three halo;

R² in each occurrence is independently selected from hydrogen, halo, andC₁₋₆alkyl;

R³ is selected from H and C₁₋₆alkyl;

R⁶ is H or C₁₋₆ alkyl;

R⁷ is H or C₁₋₆ alkyl, and R⁸ is a bond or C₁-C₃ alkylene, wherein theC₁-C₃ alkylene is optionally substituted with 1-3 substituents selectedfrom R¹⁰, or

—R⁸—OR⁷ is absent;

R⁹ in each occurrence is independently OH, halo, C₁-C₆ alkyl, C₁-C₆alkyl-OH, C₁-C₆ fluoroalkyl, C₁-C₆ heteroalkyl, C₃-C₁₀ cycloalkyl, aC₃-C₁₀ heterocycloalkyl, aryl, heteroaryl, —C₁-C₄ alkyl-(C₃-C₁₀cycloalkyl), —C₁-C₄ alkyl-(C₃-C₁₀ heterocycloalkyl), —C₁-C₄alkyl-(aryl), or —C₁-C₄ alkyl-(hetero aryl), and wherein the alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring isoptionally substituted with 1-5 substituents selected from R¹⁰;

R¹⁰ is independently selected at each occurrence from the groupconsisting of halo, —CN, —NO₂, —C₁-C₆ alkyl, —C₁-C₆ alkoxy, —C₁-C₆fluoroalkyl, —C₁-C₆ heteroalkyl, and C(O)—C₁-C₆ alkyl;

w is 0, 1, 2, or 3;

m is 0, 1, 2, or 3; and

z is 0, 1, 2, or 3.

In one embodiment, the compound of Formula XI is a compound of FormulaXII:

or a pharmaceutically acceptable salt thereof,

wherein:

Ring A is selected from:

and Q is CH or N, or

Ring A is

and Q is CH₂ or NR³.

It will be appreciated that the description of the present inventionherein should be construed in congruity with the laws and principals ofchemical bonding. In some instances it will be necessary to remove ahydrogen atom in order to accommodate a substituent at any givenlocation.

It is noted for the generic structures described herein that rings thatare substituted by two or more variables (R groups, RO groups, etc.) canindicate, for example, either vicinal (e.g., compound 1290) or geminal(e.g., compound 1302) substitution patterns.

Preferred embodiments of Formulas I-VI, including pharmaceuticallyacceptable salts thereof, as well as enantiomers, stereoisomers,rotamers, tautomers, diastereomers, atropisomers or racemates thereof,and are shown below in Table 1 and are also considered to be “compoundsof the invention.”

TABLE 1 Compound Structure ID ¹H NMR MS(M + H)⁺

 948

 949 1H NMR (400 MHz, MeOD-d4) 8.11 (s, 1H), 8.09-8.05 (m, 1H), 7.92 (s,1H), 7.76-7.63 (m, 2H), 7.33 (t, J = 8.8 Hz, 1H), 7.04 (s, 1H),3.71-3.62 (m, 1H), 3.51-3.41 (m, 2H), 2.91- 2.73 (m, 2H), 2.01-1.91 (m,2H), 1.67-1.57 (m, 2H). 452

 950

 951

1287 454

1288 436

1289

1290 482

1291 1H NMR (400 MHz, MeOD-d4) 8.08 (s, 1H), 7.89 (s, 1H), 7.71-7.65 (m,3H), 7.00 (s, 1H), 4.01-3.77 (m, 3H), 3.62-3.31 (m, 2H), 2.57-2.26 (m,2H), 1.81-1.45 (m, 3H) 484

1292 1H NMR (400 MHz, MeOD-d4) 8.07 (s, 1H), 7.97-7.75 (m, 2H), 7.70 (s,1H), 7.51-7.26 (m, 2H), 6.99 (s, 1H), 4.02-3.76 (m, 3H), 3.61-3.30 (m,2H), 2.51-2.30 (m, 2H), 1.83-1.42 (m, 3H) 466

1293

1294 456

1295 1H NMR (400 MHz, MeOD-d4) 8.14 (s, 1H), 7.96 (s, 1H), 7.72-7.63 (m,3H), 6.99 (s, 1H), 4.91-4.77 (m, 1H), 4.31-4.17 (m, 1H), 3.72-3.39 (m,4H). 458

1296 1H NMR (400 MHz, MeOD-d4) 8.14 (s, 1H), 7.95 (s, 1H), 7.93-7.81 (m,1H), 7.69 (s, 1H), 7.57-7.39 (m, 1H), 7.31-7.22 (m, 1H), 6.99 (s, 1H),4.93- 4.77 (m, 1H), 4.33-4.19 (m, 1H), 3.71-3.35 (m, 4H). 440

1297

1298 424

1299 426

1300 1H NMR (400 MHz, MeOD-d4) 8.13 (s, 1H), 7.97-7.83 (m, 2H), 7.73 (s,1H), 7.51-7.42 (m, 1H), 7.37-7.26 (m, 1H), 7.02 (s, 1H), 4.48-4.31 (m,1H), 4.05-3.92 (m, 2H), 3.63-3.51 (m, 2H). 408

1301

1302 482

1303 484

1304 1H NMR (400 MHz, MeOD-d4) 8.07 (s, 1H), 7.95-7.82 (m, 2H), 7.70 (s,1H), 7.52-7.26 (m, 2H), 6.99 (s, 1H), 3.66-3.59 (m, 2H), 3.33 (s, 2H),2.74- 2.61 (m, 2H), 1.83-1.51 (m, 4H) 466

1305

1472 453

1473

1474

1475

1476

1477

1478

1479

1480

1481

1482

1483

1484

1485

1486

1487

1488

1489

1490

1491

1492

1493

1494

1495

1496

1497

1498

1499

1500

1501

1502

1503

1504

1505

1506

1507

1508 453

1533 498

1534 1H NMR (400 MHz, MeOD-d4) 8.07 (s, 1H), 8.03 (s, 1H), 7.89 (s, 1H),7.72- 7.47 (m, 2H), 7.28 (t, J = 8.8 HZ, 1H), 7.00 (s, 1H), 3.65-3.57(m, 2H), 3.33 (s, 3H), 3.18 (s, 2H), 2.81-2.63 (m, 2H), 1.81-1.61 (m,4H) 496

1535 480

1536 498

1537 1H NMR (400 MHz, MeOD-d4) 8.07 (s, 1H), 8.03 (s, 1H), 7.89 (s, 1H),7.72- 7.67 (m, 2H), 7.28 (t, J = 8.8 HZ, 1H), 6.99 (s, 1H), 3.63-3.31(m, 5H), 3.18 (s, 2H), 2.75-2.63 (m, 2H), 1.81-1.57 (m, 4H) 496

1538 480

1569

1571

1572

1573

1574

1575

Preparation of the Compounds of the Invention

The compounds of the invention may possess one or more stereocenters,and each stereocenter may exist independently in either the R or Sconfiguration. In one embodiment, compounds described herein are presentin optically active or racemic forms. It is to be understood that thecompounds described herein encompass racemic, optically-active,regioisomeric and stereoisomeric forms, or combinations thereof thatpossess the therapeutically useful properties described herein.

Preparation of optically active forms is achieved in any suitablemanner, including by way of non-limiting example, by resolution of theracemic form with recrystallization techniques, synthesis fromoptically-active starting materials, chiral synthesis, orchromatographic separation using a chiral stationary phase. In oneembodiment, a mixture of one or more isomer is utilized as thetherapeutic compound described herein. In another embodiment, compoundsdescribed herein contain one or more chiral centers. These compounds areprepared by any means, including stereoselective synthesis,enantioselective synthesis and/or separation of a mixture of enantiomersand/or diastereomers. Resolution of compounds and isomers thereof isachieved by any means including, by way of non-limiting example,chemical processes, enzymatic processes, fractional crystallization,distillation, and chromatography.

The methods and formulations described herein include the use ofN-oxides (if appropriate), crystalline forms (also known as polymorphs),solvates, amorphous phases, and/or pharmaceutically acceptable salts ofcompounds having the structure of any compound of the invention, as wellas metabolites and active metabolites of these compounds having the sametype of activity. Solvates include water, ether (e.g., tethrahydrofuran,methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetatesand the like. In one embodiment, the compounds described herein exist insolvated forms with pharmaceutically acceptable solvents such as water,and ethanol. In another embodiment, the compounds described herein existin unsolvated form. In one embodiment, the compounds of the inventionmay exist as tautomers. All tautomers are included within the scope ofthe compounds presented herein.

In one embodiment, compounds described herein are prepared as prodrugs.A “prodrug” refers to an agent that is converted into the parent drug invivo. In one embodiment, upon in vivo administration, a prodrug ischemically converted to the biologically, pharmaceutically ortherapeutically active form of the compound. In another embodiment, apro drug is enzymatically metabolized by one or more steps or processesto the biologically, pharmaceutically or therapeutically active form ofthe compound.

In one embodiment, sites on, for example, the aromatic ring portion ofcompounds of the invention are susceptible to various metabolicreactions. Incorporation of appropriate substituents on the aromaticring structures may reduce, minimize or eliminate this metabolicpathway. In one embodiment, the appropriate substituent to decrease oreliminate the susceptibility of the aromatic ring to metabolic reactionsis, by way of example only, a deuterium, a halogen, or an alkyl group.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In another embodiment, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet anotherembodiment, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In one embodiment, the compounds described herein are labeled by othermeans, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds havingdifferent substituents are synthesized using techniques and materialsdescribed herein and as described, for example, in Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive OrganicTransformations (VCH Publishers Inc., 1989), March, Advanced OrganicChemistry 4^(th) Ed., (Wiley 1992); Carey and Sundberg, Advanced OrganicChemistry 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green andWuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (allof which are incorporated by reference for such disclosure). Generalmethods for the preparation of compound as described herein are modifiedby the use of appropriate reagents and conditions, for the introductionof the various moieties found in the formula as provided herein.

Compounds described herein are synthesized using any suitable proceduresstarting from compounds that are available from commercial sources, orare prepared using procedures described herein.

In one embodiment, reactive functional groups, such as hydroxyl, amino,imino, thio or carboxy groups, are protected in order to avoid theirunwanted participation in reactions. Protecting groups are used to blocksome or all of the reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In another embodiment, each protective group is removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval.

Protecting groups, plus a detailed description of techniques applicableto the creation of protecting groups and their removal are described inGreene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., JohnWiley & Sons, New York, N.Y., 1999, and Kocienski, Protective Groups,Thieme Verlag, New York, N.Y., 1994, which are incorporated herein byreference for such disclosure.

Assays

HBV Capsid Protein Assembly Testing

The fluorescence quenching in vitro assembly HBV assay was developedaccording to a method described by Zlotnick and coworkers (NatureBiotechnology 2006, 24:358). The assay is based on the observation thatthe C-termini of the HBV core protein cluster together during capsidformation. This assay utilizes a mutant C150 HBV capsid protein whereall wild-type cysteines are mutated to alanines, but a C-terminalcysteine residue is preserved and is labeled with fluorescent BoDIPY-FLdye. HBV C150Bo protein is highly fluorescent, however the fluorescenceis drastically reduced during the capsid assembly process. Thus, theassay measures the ability and potency of test compounds to modulatecapsid assembly by monitoring the fluorescence of the labeled capsidC150Bo protein.

In a typical assay, the mutant HBV C150 protein (amino acids 1-150,C49A, C61A, C107A, 150C) is cloned into a T7 RNA-polymerase basedexpression vector, expressed in E. coli and purified to homogeneity as adimer. The purified HBV core protein is desalted and labeled withBODIPY-FL Dye.

In a non-limiting embodiment, the assembly assay is conducted in 96-wellplate format. The assembly reactions are carried out in 50 mM Hepesbuffer, pH 7.5 and 150 mM NaCl. The compounds are pre-incubated with theHBV CA protein for 15 min, and the assembly reactions are initiated byaddition of NaCl. The reaction is allowed to continue for 1 hour at roomtemperature.

To determine the effect on capsid assembly, each test compound isinitially screened at 4 different concentrations: 10 μM, 3 μM, 1 μM and0.3 μM in duplicates. Primary hits are compounds that show activity inthe assembly assay with EC₅₀ less than 10 uM and a representative groupof these active compounds is shown in Table 2. Identified primary hitsare confirmed in follow-up studies as described elsewhere herein. Knownmodulators of HBV CA assembly, such as HAP-1 and BAY 41-4109, are usedas control compounds in these experiments and exhibited EC₅₀ valuesconsistent with the literature. EC₅₀ values for test compounds aredetermined via analysis of the dose-response curve.

HBV Antiviral Testing

Compounds active in the HBV assembly assay are tested for their activityand toxicity in cellular assay. In the first anti-viral assay, theability of compounds to inhibit HBV replication in an HBV-producinghepatoma cell line using the dot-blot method is evaluated.

Briefly, confluent monolayers of HepG2-2.2.15 cells are incubated withcomplete medium containing various concentrations of a test compound.Three days later, the culture medium is replaced with fresh mediumcontaining the appropriately diluted test compound. Six days followingthe initial administration of the test compound, the cell culturesupernatant is collected, and cell lysis is performed. The samples areapplied onto Nylos membranes and DNA is immobilized to the membrane byUV cross-linking. After pre-hybridization, the HBV probe is added andthe hybridization is performed overnight. The membranes are exposed tothe Kodak films; antiviral activity is calculated from the reduction inHBV DNA levels (EC₅₀). The EC₅₀ for antiviral activity is calculatedfrom the dose response curves of active compounds. Assay performanceover time is monitored by the use of the standard positive controlcompounds ETV, BAY 41-4109, and HAP-1.

Compound cytotoxity (TC₅₀) is measured in this same HepG2-2.2.15 cellline using a CellTiter Blue-based cytotoxicity assay employed asrecommended by manufacturer (Promega). To confirm and expand theseresults, a second antiviral assay is carried out on active compoundsusing the stable HBV cell line HepG2.2.15 and measuring anti-HBV potencyby real-time PCR and cytotoxicity by CellTiter Blue. In this assay, 24hours after cell seeding, HepG2-2.2.15 cells are incubated with completemedium containing various concentrations of a test compound with BAY41-4109 and HAP-1 used as positive controls. After three days, theculture medium is replaced with fresh medium containing theappropriately diluted test compound. The cell culture is collected sixdays following the initial administration of the test compound, followedby HBV DNA extraction using QIAamp 96 DNA Blood Kit (Qiagen). Theextracted HBV DNA is diluted and analyzed by Real-Time PCR. A standardcurve is generated by plotting Ct value vs the amount of HBV plasmidstandard. Cytotoxicity is determined similarly to the above describedmethod by applying a dye uptake method (CellTiter Blue kit, Promega).

Prevention of HBV Pre-Genomic RNA (pgRNA) Incorporation

The anti-viral activity of the compounds of the invention is assessedbased on their ability to suppress both extracellular and intracellularHBV DNA production in two different cell culture models of HBVreplication. To assess if these effects are due to disruption ofintracellular capsid assembly, a particle-gel assay that allowsquantitation of intracellular viral capsids, as well as encapsidatedpre-genomic RNA and DNA, is performed. The assay relies on agarose gelseparation of viral capsid from free capsid/core subunits and viralpg-RNA and DNA.

Methods of Treatment

The invention includes a method of treatment of an HBV infection in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of a compound of the invention.

The invention also includes a method of reducing viral load associatedwith an HBV infection in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of acompound of the invention.

The invention further includes a method of reducing reoccurrence of anHBV infection in an individual in need thereof, comprising administeringto the individual a therapeutically effective amount of a compound ofthe invention.

The invention also includes a method of reducing an adversephysiological impact of an HBV infection in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of a compound of the invention.

The invention includes a method of reducing, slowing, or inhibiting anHBV infection in an individual in need thereof, comprising administeringto the individual a therapeutically effective amount of a compound ofthe invention.

The invention also includes a method of inducing remission of hepaticinjury from an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of the invention.

The invention further includes a method of reducing the physiologicalimpact of long-term antiviral therapy for HBV infection in an individualin need thereof, comprising administering to the individual atherapeutically effective amount of a compound of the invention.

The invention also includes a method of eradicating an HBV infection inan individual in need thereof, comprising administering to theindividual a therapeutically effective amount of a compound of theinvention.

The invention further includes a method of prophylactically treating anHBV infection in an individual in need thereof, wherein the individualis afflicted with a latent HBV infection, comprising administering tothe individual a therapeutically effective amount of a compound of theinvention.

In one embodiment, the methods described herein further compriseadministering at least one therapeutic agent selected from the groupconsisting of nucleotide/nucleoside analogs, entry inhibitors, fusioninhibitors, interferon agents, and any combination of those or otherantiviral mechanisms. In another embodiment, the compound of theinvention and the at least one additional therapeutic agent areco-formulated. In yet another embodiment, the compound of the inventionand the at least one additional therapeutic agent are co-administered.

In one embodiment, the individual is refractory to other therapeuticclasses of HBV drugs (e.g, HBV polymerase inhibitors, interferons, viralentry inhibitors, viral maturation inhibitors, distinct capsid assemblymodulators, antiviral compounds of distinct or unknown mechanism, andthe like, or combinations thereof). In another embodiment, the method ofthe invention reduces viral load in an individual suffering from an HBVinfection to a greater extent compared to the extent that othertherapeutic classes of HBV drugs reduce viral load in the individual.

In one embodiment, the method of the invention reduces viral load in anindividual suffering from an HBV infection, thus allowing lower doses orvarying regimens of combination therapies to be used.

In one embodiment, the method of the invention causes a lower incidenceof viral mutation and/or viral resistance compared to other classes ofHBV drugs, thereby allowing for long term therapy and minimizing theneed for changes in treatment regimens.

In one embodiment, the method of the invention increases theseroconversion rate beyond that of current treatment regimens.

In one embodiment, the method of the invention increases and/ornormalizes and/or restores normal health, elicits full recovery ofnormal health, restores life expectancy, and/or resolves the viralinfection in the individual in need thereof.

In one embodiment, the method of the invention eradicates HBV from anindividual infected with HBV, thereby obviating the need for long termand/or life-long treatment, or shortening the duration of treatment,and/or allowing for reduction in dosing of other antiviral agents.

Accordingly, in one embodiment, provided herein is a method of treatingan HBV infection in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of acompound of Formula I, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula II, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula III, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula IV, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula V, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula VI, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula VII, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula VIII, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula IX, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula X, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula XI, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula XII, or a pharmaceutically acceptable salt thereof.

Accordingly, in one embodiment, provided herein is a method of treatingan HBV infection in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of acompound of Table 1, or a pharmaceutically acceptable salt thereof.

Combination Therapies

The compounds of the present invention are intended to be useful incombination with one or more additional compounds useful for treatingHBV infection. These additional compounds may comprise compounds of thepresent invention or compounds known to treat, prevent, or reduce thesymptoms or effects of HBV infection. Such compounds include but are notlimited to HBV polymerase inhibitors, interferons, pegylatedinterferons, viral entry inhibitors, viral maturation inhibitors,literature-described capsid assembly modulators, and other agents withdistinct or unknown mechanisms that affect the HBV life cycle and/oraffect the consequences of HBV infection.

In non-limiting examples, the compounds of the invention may be used incombination with one or more drugs (or a salt, solvate or prodrugthereof) selected from the group consisting of:

HBV reverse transcriptase inhibitors, and DNA and RNA polymeraseinhibitors, including but are not limited to: lamivudine (3TC, Zeffix,Heptovir, Epivir, and Epivir-HBV), entecavir (Baraclude, Entavir),adefovir dipivoxil (Hepsara, Preveon, bis-POM PMEA), tenofovirdisoproxil fumarate (Viread, TDF or PMPA);

Interferons and pegylated interferons, including but not limited tointerferon alpha (IFN-α), interferon lambda (IFN-λ), and interferongamma (IFN-γ);

viral entry inhibitors;

viral maturation inhibitors;

literature-described capsid assembly modulators, such as but not limitedto BAY 41-4109;

compounds of distinct or unknown mechanism, such as but not limited toAT-61((E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide),AT-130((E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide),and similar analogs.

In one embodiment, the additional therapeutic agent is a pegylatedinterferon alpha drug, including, but not limited to, PEGASYS®.

In another embodiment, the additional therapeutic agent selected fromimmune modulator or immune stimulator therapies, which includesbiological agents belonging to the interferon class, such as interferonalpha 2a or 2b or modified interferons such as pegylated interferon,alpha 2a, alpha 2b, lamda; or TLR modulators such as TLR-7 agonists orTLR-9 agonists, or antiviral agents that block viral entry or maturationor target the HBV polymerase such as nucleoside or nucleotide ornon-nucleos(t)ide polymerase inhibitors, and agents of distinct orunknown mechanism including agents that disrupt the function of otheressential viral protein(s) or host proteins required for HBV replicationor persistence.

In an embodiment of the combination therapy, the reverse transcriptaseinhibitor and/or DNA and/or RNA polymerase inhibitor Zidovudine,Didanosine, Zalcitabine, ddA, Stavudine, Lamivudine, Abacavir,Emtricitabine, Entecavir, Apricitabine, Atevirapine, ribavirin,acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir,Tenofovir, Adefovir, PMPA, cidofovir, Efavirenz, Nevirapine,Delavirdine, or Etravirine.

In another embodiment of the combination therapy, the TLR-7 agonist isselected from the group consisting of SM360320(9-benzyl-8-hydroxy-2-(2-methoxy-ethoxy)adenine) and AZD 8848 (methyl[3-({[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl][3-(4-morpholinyl)propyl]amino}methyl)phenyl]acetate).

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22: 27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the patienteither prior to or after the onset of a HBV infection. Further, severaldivided dosages, as well as staggered dosages may be administered dailyor sequentially, or the dose may be continuously infused, or may be abolus injection. Further, the dosages of the therapeutic formulationsmay be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat HBV infection in the patient. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the state of the disease or disorder in thepatient; the age, sex, and weight of the patient; and the ability of thetherapeutic compound to treat HBV infection in the patient. Dosageregimens may be adjusted to provide the optimum therapeutic response.For example, several divided doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. A non-limiting example of an effective dose rangefor a therapeutic compound of the invention is from about 1 and 5,000mg/kg of body weight/per day. One of ordinary skill in the art would beable to study the relevant factors and make the determination regardingthe effective amount of the therapeutic compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of HBV infection in a patient.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,in the composition. Prolonged absorption of the injectable compositionsmay be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate or gelatin. In oneembodiment, the pharmaceutically acceptable carrier is not DMSO alone.

In one embodiment, the compositions of the invention are administered tothe patient in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the patient in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary fromindividual to individual depending on many factors including, but notlimited to, age, disease or disorder to be treated, gender, overallhealth, and other factors. Thus, the invention should not be construedto be limited to any particular dosage regime and the precise dosage andcomposition to be administered to any patient will be determined by theattending physical taking all other factors about the patient intoaccount.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., a drug used fortreating Parkinson's Disease) as described herein is less than about1,000 mg, or less than about 800 mg, or less than about 600 mg, or lessthan about 500 mg, or less than about 400 mg, or less than about 300 mg,or less than about 200 mg, or less than about 100 mg, or less than about50 mg, or less than about 40 mg, or less than about 30 mg, or less thanabout 25 mg, or less than about 20 mg, or less than about 15 mg, or lessthan about 10 mg, or less than about 5 mg, or less than about 2 mg, orless than about 1 mg, or less than about 0.5 mg, and any and all wholeor partial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof HBV infection in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

Examples

The representative synthesis procedures provided below can be used toprepare the compounds of Formula I, e.g., the compounds of Table I.These examples are provided for the purpose of illustration only, andthe invention is not limited to these examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Unless otherwise noted, all starting materials and resins were obtainedfrom commercial suppliers and used without purification.

1. Preparation of Amine 1 and Amine 2

1.1 Preparation of Compound 2

To a solution of Me₃SOI (17.5 g, 0.079 mol) in DMSO (80 mL) was addedNaH (3.4 g, 0.085 mol) at 0° C. The resulting mixture was stirred atroom temperature for 1 hour, then Bu₄NBr (1.61 g, 0.005 mol) was added.A solution of Compound 1 (10.0 g, 0.053 mol) in DMSO (40 mL) was addeddropwise slowly into the mixture and stirred at room temperature for 2hour. The mixture was poured into ice water and extracted with EA. Thecombined organic phases were washed with brine, dried over Na₂SO₄, andconcentrated in vacuum to give the desired product (9.8 g, 91.08%).

1.2 Preparation of Compound 3

To a solution of Compound 2 (3.0 g, 14.78 mmol) in MeOH (60 mL) wasadded BF₃Et₂O (2.52 g, 17.73 mmol) at room temperature. The mixturestirred at room temperature for 12 hours. The reaction was quenched byNaHCO₃ solution. The mixture was extracted with EtOAc. The organic layerwas concentrated to give the desired Compound 3, and used directly inthe next step without further purification (3.2 g, 98%).

1.3 Preparation of Amine 1

To a solution of Compound 3 (2.3 g, 9.78 mmol) in MeOH was addedPd(OH)₂/C (1 g), and the resulting mixture was stirred under H₂ balloonatmosphere for 12 hours at 25° C. The mixture was filtered and thefiltrate was concentrated to give the desired product as colorless oil(1.3 g, 92%).

1.4 Preparation of Compound 4

To a solution of Compound 2 (3.0 g, 14.78 mmol) in MeOH (30 mL) wasadded CH₃ONa (2.4 g, 45 mmol) at 0° C. The mixture was stirred at 25° C.for 12 hours. The reaction was quenched with NH₄Cl (100 mL) solution,and the mixture was extracted with EtOAc (100 mL). The organic layer wasconcentrated to give the desired product, and used directly in the nextstep without further purification (3.2 g, 98%).

1.5 Preparation of Amine 2

To a solution of Compound 4 (2.3 g, 9.78 mmol) in MeOH was addedPd(OH)₂/C (1 g), and the resulting mixture was stirred under H₂ balloonatmosphere for 12 hours at 25° C. The mixture was filtered and thefiltrate was concentrated to give the desired product as colorless oil(1.3 g, 92%).

2. Preparation of Compound 949

2.1 Preparation of Compound 2

To a solution of Compound 1 (2.5 g, 10 mmol) and in DMF (30 mL) wasadded NaH (600 mg, 15 mmol) at 0° C., the mixture was stirred at 0° C.for 30 min. Then SEMCl (2.49 g, 15 mmol) was added dropwised at 0° C.and stirred at room temperature for 2 hours. The mixture was dilutedwith EA (100 mL) and water (50 mL). The organic layer was washed withsaturated NH₄Cl (100 mL*3), dried over Na₂SO₄, and concentrated in vacuoto give Compound 2 as yellow solid (3.7 g, 98%). LCMS: 384/386 [M+1].

2.2 Preparation of Compound 3

A mixture of Compound 2 (1.92 g, 5 mmol), (4-methoxyphenyl) methanethiol(0.69 g, 5.5 mmol), Pd₂(dba)₃ (0.23 g, 0.25 mol), Xantphos (0.29 g, 0.5mmol) and DIPEA (1.29 g, 10 mmol) in dioxane (50 mL) was heated toreflux overnight under N₂ atmosphere. The mixture was filtered and thefiltrate was concentrated in vacuo. The residue was purified by silicagel chromatography (PE:EA=10:1) to give Compound 3 as yellow solid (2.0g, 88%). LCMS: 458 [M+1].

2.3 Preparation of Compound 4

To a stirred solution of Compound 3 (2.2 g, 5 mmol) in THF (20 mL) wasadded aqueous LiOH (5 mL, 3 M) solution. The mixture was heated toreflux for 2 hours. After cooled to room temperature, the mixture wasadjusted pH to 4 with 2 M HCl. The mixture was extracted with EA (100mL*2). The organic layers was dried over Na₂SO₄ and concentrated to givedesired Compound 4 as yellow solid (2.1 g, 99%). LCMS: 444 [M+1].

2.4 Preparation of Compound 5

To a solution of Compound 4 (2.2 g, 5 mmol) and 3-chloro-4-fluoroaniline(870 mg, 6 mmol) in DMF (30 mL) was added HATU (2.8 g, 7.5 mmol) andDIEA (967 mg, 7.5 mmol) at room temperature, then the mixture wasstirred at 60° C. for 12 h. The mixture was diluted with EA (100 mL).The organic layer was washed by saturated NH₄Cl (100 mL*3), dried overNa₂SO₄, and concentrated in vacuo. The residue was purified bychromatography to give Compound 5 as yellow solid (2.0 g, 70%). LCMS:571 [M+1].

2.5 Preparation of Compound 6

To a suspension of Compound 5 (470 mg, 1 mmol) in MeCN/AcOH/H₂O (80/1/2,5 mL) at −15° C. was added1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione (290 mg, 1.5 mmol), themixture was stirred at −15° C. for 8 hours. The residue was used in thenext step directly. LCMS: 517/519 [M+1].

2.6 Preparation of Compound 7

To a mixture of Compound 6 (1 mmol, from the last step) was addedpiperidin-4-ol (202 mg, 2 mmol) and TEA (400 mg, 4 mmol), the mixturewas stirred at room temperature for 1 hour. The solvent was removed andthe residue was dissolved in EA (50 mL), the organic layer was washedwith saturated NH₄Cl (50 mL*2), then dried over Na₂SO₄ and concentratedto give crude Compound 7 which was used for the next step withoutfurther purification. LCMS: 582 [M+1].

2.7 Preparation of Compound 949

To a solution of Compound 7 (1 mmol, from the last step) in THF (10 mL)was added TBAF (1050 mg, 4 mmol) and ethane-1,2-diamine (240 mg, 4mmol), the mixture was stirred at 70° C. for 30 hours. The solvent wasremoved and the residue was purified by pre-HPLC (FA) to give desiredCompound 949 (95 mg, yield 21.1% for the three steps) as white solid. ¹HNMR (400 MHz, MeOD-d4) 8.11 (s, 1H), 8.09-8.05 (m, 1H), 7.92 (s, 1H),7.76-7.63 (m, 2H), 7.33 (t, J=8.8 Hz, 1H), 7.04 (s, 1H), 3.71-3.62 (m,1H), 3.51-3.41 (m, 2H), 2.91-2.73 (m, 2H), 2.01-1.91 (m, 2H), 1.67-1.57(m, 2H). LCMS: 452 [M+1].

3. Preparation of Compound 1472

3.1 Preparation of Compound 2

To HSO₃Cl (80 mL) was added Compound 1 (20.0 g, 0.13 mol) portionwise at0° C., then the resulting mixture was heated to 140° C. for 4 hours.After cooled to room temperature, the mixture was poured into ice-water.The resulting precipitate was collected by filtration and dried to givedesired Compound 2 (25.0 g, 76%) as white solid.

3.2 Preparation of Compound 3

Compound 2 (6.0 g, 23 mmol) was added to a mixture of HNO₃ (8 mL) inH₂SO₄ (40 mL), and heated to 90° C. for 4 hours. After cooled to roomtemperature, the mixture was added to ice-water slowly. The resultingprecipitate was collected by filtration and dried to give desiredCompound 3 (2.6 g, 38%) as white solid. ¹H-NMR (CDCl₃, 400 MHz): δ ppm9.05 (d, J=2.0 Hz, 1H), 8.76 (d, J=2.0 Hz, 1H).

3.3 Preparation of Compound 4

A mixture of Compound 3 (0.6 g, 2 mmol) and SOCl₂ (10 mL) was heated to90° C. for 4 hours. The mixture was concentrated in vacuo. The residuewas dissolved with toluene (10 mL) and heated to 90° C.3-Chloro-4-fluoroaniline (290 mg, 2 mmol) was added and the mixture wascontinued to heat to reflux for 4 hours. The mixture was concentrated togive desired Compound 4 (0.8 g, crude) as yellow solid, which was usedfor the next step without further purification.

3.4 Preparation of Compound 6

To a mixture of Compound 4 (0.8 g, crude) and piperidin-4-ol (202 mg, 2mmol) in CH₃CN (10 mL), was added Et₃N (202 mg, 2 mmol), and stirred atroom temperature for 4 hours. The mixture was diluted with EA (50 mL),the organic layer was washed with NH₄Cl (50 mL*2) and concentrated togive desired Compound 6 (0.5 g, crude) as yellow solid, which was usedfor the next step without further purification.

3.5 Preparation of Compound 7

To a solution of Compound 6 (0.5 g, crude) in MeOH (10 mL) was addedammonia (2 mL, 28%), and the mixture was heated to 70° C. for 4 hours.The mixture was concentrated in vacuo to give desired Compound 7 (0.5 g,crude) as yellow solid, which was used for the next step withoutpurification.

3.6 Preparation of Compound 8

A mixture of Compound 7 (0.5 g, crude), Fe (400 mg, 7 mmol) and NH₄Cl(400 mg, 7 mmol) in MeOH—H₂O (10 mL/2 mL) was heated to 70° C. for 4hours. The mixture was filtered and washed with EA (50 mL). The filtratewas washed with saturated NH₄Cl (50 mL*2) and concentrated to givedesired Compound 8 (0.3 g, crude) as white solid, which was used for thenext step without purification.

3.7 Preparation of Compound 1472

A mixture of Compound 8 (0.3 g, crude) in formic acid (10 mL) wasmicrowaved to 110° C. for 10 min. The mixture was concentrated in vacuo.The residue was dissolved in MeOH—H₂O (10 mL/2 mL), and K₂CO₃ (270 mg, 2mmol) was added. The mixture was heated to 80° C. for 2 hours. AfterLCMS showed the reaction was finished, the mixture was concentrated invacuo and extracted with EA. The organic phase was concentrated in vacuoand the residue was purified via acid preparative HPLC to give Compound1472 (35 mg, yield 3.6% over the seven steps) as white solid.

HBV Assembly Assay

Selected compounds of the invention were assayed in the HBV assemblyassay, as described elsewhere herein. The assembly assay was conductedin 96-well plate format. The assembly reactions were carried out in 50mM Hepes buffer, pH 7.5 and 150 mM NaCl. The compounds werepre-incubated with the HBV CA protein for 15 min, and the assemblyreactions were initiated by addition of NaCl. The reaction was allowedto continue for 1 hour at room temperature. The 96-well plate assemblyassay consistently had Z′ factors greater than 0.7 and were robust andreproducible both from plate-to-plate and day-to-day.

To determine the effect on capsid assembly, each test compound wasinitially screened at 4 different concentrations: 10 μM, 3 μM, 1 μM and0.3 μM in duplicates. Primary hits were compounds that show activity inthe assembly assay with EC₅₀ less than 10 μM and a representative groupof these active compounds is illustrated in Table 2.

TABLE 2 “Activity” represents activity in HBV assembly assay (‘+’indicates EC₅₀ < 10 μM) Compound Activity Compound Activity 949 + 1287 +1291 + 1292 + 1294 + 1296 + 1299 + 1300 + 1303 + 1304 + 1470 + 1508 +1533 + 1534 + 1535 + 1536 +

Dot-Blot Assay

Selected compounds, which were shown to be active in the HBV assemblyassay, were tested for their activity and toxicity in cellular assay. Inthe first anti-viral assay, the ability of compounds to inhibit HBVreplication in an HBV-producing hepatoma cell line using the dot-blotmethod was evaluated.

Confluent monolayers of HepG2-2.2.15 cells were incubated with completemedium containing various concentrations of a test compound. Three dayslater, the culture medium was replaced with fresh medium containing theappropriately diluted test compound. Six days following the initialadministration of the test compound, the cell culture supernatant wascollected, and cell lysis was performed. The samples were applied ontoNylos membranes and DNA was immobilized to the membrane by UVcross-linking. After pre-hybridization, the HBV probe was added and thehybridization was performed overnight. The membranes were exposed to theKodak films; antiviral activity was calculated from the reduction in HBVDNA levels (EC₅₀). The EC₅₀ for antiviral activity was calculated fromthe dose response curves of active compounds. Assay performance overtime was monitored by the use of the standard positive control compoundsETV, BAY 41-4109, and HAP-1. Results are illustrated in Table 3.

Cytotoxity (CC₅₀) was measured in this same HepG2-2.2.15 cell line usinga CellTiter Blue-based cytotoxicity assay employed as recommended bymanufacturer (Promega). All compounds in Table 3 demonstrated lowtoxicity at 5 μM.

TABLE 3 “Activity” represents activity in dot-blot-assay (‘+’ indicatesEC₅₀ < 10 μM) Compound Activity Compound Activity 949 + 1288 + 1290 +1294 + 1298 + 1300 + 1302 + 1472 + 1508 + 1533 + 1535 + 1537 +

Prevention of HBV Pre-Genomic RNA (pgRNA) Incorporation

The compounds of the invention were assessed for their ability tosuppress both extracellular and intracellular HBV DNA production in twodifferent cell culture models of HBV replication. A particle-gel assaythat allows quantitation of intracellular viral capsids, as well asencapsidated pre-genomic RNA and DNA, was performed. The assay relied onagarose gel separation of viral capsid from free capsid/core subunitsand viral pg-RNA and DNA.

This assay revealed that the compounds of the invention preventpackaging of pre-genomic RNA into the viral capsid without significanteffect on intracellular core particle levels. This effect is consistentwith the biochemical activity of the compounds of the invention, whichact as allosteric effectors that misdirect in vitro assembly leading toformation of aberrant, non-functional particles. The potent antiviraleffect is due to that pg-RNA encapsidation is required for viral DNAsynthesis.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A compound selected from the group consisting of:

or pharmaceutically acceptable salts thereof. 2-17. (canceled)
 18. Amethod of treating an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound according to claim
 1. 19. A method of reducing theviral load associated with an HBV infection in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of a compound according to claim
 1. 20. A method ofreducing reoccurrence of an HBV infection in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of a compound according to claim
 1. 21. A method ofreducing an adverse physiological impact of an HBV infection in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of a compound according to claim
 1. 22.A method of inducing remission of hepatic injury from an HBV infectionin an individual in need thereof, comprising administering to theindividual a therapeutically effective amount of a compound according toclaim
 1. 23. A method of reducing the physiological impact of long-termantiviral therapy for HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound according to claim
 1. 24. A method ofprophylactically treating an HBV infection in an individual in needthereof, wherein the individual is afflicted with a latent HBVinfection, comprising administering to the individual a therapeuticallyeffective amount of a compound according to claim
 1. 25. The method ofclaim 1, further comprising administering to the individual at least oneadditional therapeutic agent selected from the group consisting of a HBVpolymerase inhibitor, interferon, pegylated interferon, viral entryinhibitor, viral maturation inhibitor, HAP-1, BAY 41-4109, reversetranscriptase inhibitor, a TLR-agonist,(E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide,and(E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide,and a combination thereof.
 26. The method of claim 25, wherein thepegylated interferon is pegylated interferon alpha (IFN-α), pegylatedinterferon lambda (IFN-λ), or pegylated interferon gamma (IFN-γ). 27.The method of claim 25, wherein the reverse transcriptase inhibitor isat least one of Zidovudine, Didanosine, Zalcitabine, ddA, Stavudine,Lamivudine, Abacavir, Emtricitabine, Entecavir, Apricitabine,Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir,ganciclovir, valganciclovir, Tenofovir, Adefovir, cidofovir, Efavirenz,Nevirapine, Delavirdine, or Etravirine.
 28. The method of claim 25,wherein the compound and the at least one additional therapeutic agentare co-formulated.
 29. The method of claim 25, wherein the compound andthe at least one additional therapeutic agent are co-administered. 30.The method of claim 25, wherein administering the compound allows foradministering the at least one additional therapeutic agent at a lowerdose or frequency as compared to the administering of the at least oneadditional therapeutic agent alone that is required to achieve similarresults in prophylactically treating an HBV infection in an individualin need thereof.
 31. The method of claim 25, wherein beforeadministering the therapeutically effective amount of the compound, theindividual is known to be refractory to a compound selected from thegroup consisting of a HBV polymerase inhibitor, interferon, pegylatedinterferon, viral entry inhibitor, viral maturation inhibitor, HAP-1,BAY 41-4109,(E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide,and(E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide,and combination thereof.
 32. The method of claim 25, wherein theadministering of the compound reduces viral load in the individual to agreater extent compared to the administering of a compound selected fromthe group consisting of a HBV polymerase inhibitor, interferon,pegylated interferon, viral entry inhibitor, viral maturation inhibitor,HAP-1, BAY 41-4109,(E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide,and(E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide,and combination thereof.
 33. The method of claim 25, wherein theadministering of the compound causes a lower incidence of viral mutationand/or viral resistance than the administering of a compound selectedfrom the group consisting of a HBV polymerase inhibitor, interferon,pegylated interferon, viral entry inhibitor, viral maturation inhibitor,HAP-1, BAY 41-4109,(E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide,and(E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide,and combination thereof.
 34. The method of claim 26, wherein thepegylated interferon alfa is pegylated interferon alpha-2a.